Mutant prenyl diphosphate synthase, DNA encoding mutant prenyl diphosphate synthase and process for producing mutant prenyl phosphate synthase

ABSTRACT

Manufacture or use of a mutant prenyl diphosphate synthase in which the amino acid residue located at the fifth position in the N-terminal direction from D of the N-terminal of the aspartic acid-rich domain DDXX(XX)D (the two X&#39;s in the parentheses may not be present) present in the second region among the conserved regions of the prenyl diphosphate synthase has been substituted by another amino acid.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of application Ser. No.08/886,466 filed July 1, 1997, now U.S. Pat. No. 6,040,165 which isincorporated herein by reference in its entirety, and claims prioritytherefrom.

BACKGROUND OF INVENTION

1. Field of Invention

The present invention relates to novel mutant enzymes which synthesizelinear prenyl diphosphates that are precursors of compounds, importantto organisms, such as steroids, ubiquinones, dolichols, carotenoids,prenylated proteins, animal hormones, plant hormones, and the like, or agene thereof etc.

2. Related Art

Of the substances having important functions in the body, manysubstances are biosynthesized using isoprene (2-methyl-1,3-butadiene) asa building block. These compounds are called isoprenoids, terpenoids, orterpenes, and are classified depending on the number of carbon atomsinto hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15),diterpenes (C20), sesterterpenes (C25), triterpenes (C30), tetraterpenes(C40), and the like. The actual synthesis starts with the mevalonatepathway through which mevalonic acid-5-diphosphate is synthesized,followed by the synthesis of isopentenyl diphosphate (IPP) which is anactive isoprene unit.

The identity of the isoprene unit that was proposed as an speculatedprecursor was found to be IPP, the so-called active isoprene unit.Dimethylallyl diphosphate (DMAPP), an isomer of IPP, being used as asubstrate in the reaction of isopentenyl adenine, which is known as acytokinin and is one of the plant hormones, is also known to undergocondensation reaction with IPP to synthesize linear active isoprenoidssuch as geranyl diphosphate (GP), neryl diphosphate, farnesyldiphosphate (FPP), geranylgeranyl diphosphate (GGPP), geranylfarnesyldiphosphate (GFPP), hexaprenyl diphosphate (HexPP), heptaprenyldiphosphate (HepPP), and the like.

There are Z-type and E-type condensation reactions. GPP is a product ofE-type condensation and neryl diphosphate is a product of Z-typecondensation. Although, the all-E-type is considered to be the activeform in FPP and GGPP, the Z-type condensation reactions lead to thesynthesis of various polyprenols found in natural rubber, dolichols,bactoprenols (undecaprenols), and plants. They are believed to undergothe condensation reaction using the phosphate ester bond energy of thepyrophosphate and/or the carbon backbone present in the molecule toproduce pyrophosphate and/or phosphate as the byproduct of the reaction.

FPP or GGPP serves as a reaction substrate leading to the synthesis ofprenylated proteins (from FPP or GGPP) represented by G-proteins thatare important in the mechanism of signal transduction in the cell; cellmembrane lipids (from GGPP) of archaea; squalene (from FPP) which is aprecursor of steroids; and phytoene (from GGPP) which is a precursor ofcarotenoid. Prenyl diphosphates from HexPP and HepPP having six andseven isoprene units respectively to prenyl diphosphates having tenisoprene units serve as the precursor of synthesis of ubiquinone andmenaquinone (vitamin K2) that work in the electron transport system.

Furthermore, via the biosynthesis of these active-form isoprenoids, thefollowing planty kinds of compounds that are vital to life have beensynthesized. Just to mention a few, there are plant hormones ofcytokinins and isopetenyl adenosine-modified tRNA that use hemiterpenesas their precursor for synthesis, monoterpene geraniol and the nerolisomers thereof that are the main components of rose oil perfume, and acamphor tree extract camphor which is an insecticide. Sesquiterpensinclude juvenile hormones of insects, diterpenes include a plant hormonegibberellin, trail pheromones of insects, and retinols and retinals thatfunction as the visual pigment precursors, binding components of thepurple membrane proteins of halophilic archaea, and vitamin A.

Furthermore, using squalene, a triterpene, a variety of steroidcompounds have been synthesized, including, for example, animal sexhormones, vitamin D, ecdysone which is an mating hormone of insects, aplant hormone brassinolide, and components of plasma membranes. Variouscarotenoids of tetraterpenes that are precursors of various pigments oforganisms and vitamin A are also important compounds derived from activeisoprenoids. Compounds such as hlorophyll, pheophytin, tocopherol(vitamin E), and phylloquinone (vitamin K1) are also derived fromtetraterpenes.

The active isoprenoid synthases that consecutively condense IPP withsuch allylic substrates DMAPP, GPP, FPP, GGPP, GFPP, and the like arecalled prenyl diphosphate synthases, and are also named, based on themaximum chain length of the major reaction products, for examplefarnesyl diphosphate synthase (FPP synthase), geranylgeranyl diphosphate(GGPP synthase), and the like. There are reports on purification,activity measurement, gene cloning, and its nucleotide sequencing ofenzymes such as farnesyl diphosphate synthase, geranylgeranyldiphosphate synthase, hexaprenyl diphosphate synthase, heptaprenyldiphosphate synthase, octaprenyl diphosphate synthase, nonaprenyldiphosphate synthase (solanesyl diphosphate synthase), undecaprenyldiphosphate synthase, and the like from bacteria, archaea, fungi,plants, and animals.

These active isoprenoid synthases constituting the basis of synthesis ofa great variety of compounds that are important both in the industry andin the field of life sciences have attracted little attention regardingtheir industrial applications due to their unstable character and lowspecific activities. However, with the isolation of the genes of FPPsynthase and GGPP synthase from thermophilic bacteria and archaea [A.Chen and D. Poulter (1993) J. Biol. Chem. 268: 11002-11007, T. Koyama etal. (1993) J. Biochem. 113: 355-363, S. -i, Ohnuma et al. (1994) J.Biol. Chem. 269: 14792-14797], their availability as enzymes hasincreased.

The enzymes that synthesize prenyl diphosphates having 20 to 25 carbonsare homodimers and are relatively easy to be reacted in vitro, as havebeen published in many reports. However, the enzymes that synthesizeprenyl diphosphates having chain lengths exceeding the above-mentionedlength are believed to be heterodimers, or to require additional factorssuch as a lipid, and the like. Therefore, in order to realize industrialapplication thereof, it was necessary to find optimal conditions thatpermit reassembly of two kinds of subunits or additional factors, whichwas a difficult task.

Therefore, there has been a need for the technology that enables to makethe homodimer-type thermostable prenyl diphosphate synthases capable ofsynthesizing prenyl diphosphates having a longer chain length, byartificially altering the amino acid sequence of the homodimer typeprenyl diphosphate synthases that are stable and have high specificactivity derived from a thermophilic organism.

As for the prenyl diphosphate synthases derived from thermophilicorganisms, there are at present examples of the altered FPP synthasederived from Bacillus stearothermophilus and GGPP synthase derived fromSulfolobus acidocaldarius. The mutant enzyme of FPP synthase of Bacillusstearothermophilus and the gene thereof were selected based on the colorchange of the organism by lycopene produced by coexistence of crtB (thegene of phytoene synthase) and crtI (the gene of phytoene desaturase,cis:trans isomerase) derived from Erwinia uredovora and the gene of FPPsynthase of the mutant B. stearothermophilus in Escherichia coli. GGPPsynthase and its mutant and the gene thereof of S. acidocaldarius wereselected based on the activity of complementing the glycerol metabolicactivity of the HexPP synthase-deficient budding yeast of Saccharomycescereviceae.

The coexistence method of the CrtB and CrtI genes of E. uredovora cannotbe used for screening the reaction products longer than GGPP of themutant enzyme, and the screening method using the complementationactivity of the HexPP synthase-deficient budding yeast Saccharomycescereviceae cannot be used for specific detection of the reactionproducts longer than HexPP. These genetic screening methods are capableof cloning the genes of the mutant prenyl diphosphate synthases havingthe synthetic activities of GGPP, GFPP, and HexPP, but cannotsystematically control the chain length of the reaction products ofprenyl diphosphate synthases with the intention of extending the chainlength of the reaction products. A rule for that purpose is not known,either.

SUMMARY OF INVENTION

It is an object of the invention to establish a rule for systematiccontrol of the chain length of reaction products by modifying amino acidresidues of prenyl diphosphate enzymes. A new enzyme that is more stableor that has a high specific activity more adaptable to industrialapplication would make it possible to obtain immediately a mutant enzymeor the gene thereof that synthesizes prenyl diphosphate having a longerchain length by modifying amino acid residues based on the above rule.

From the information on the nucleotide sequence of the gene of GGPPsynthase of the mutant S. acidocaldarius, it was clarified that out ofthe two proposed Asp-rich domains based on the analysis of the aminoacid sequence of prenyl diphosphate synthase, the amino acid residuelocated at the fifth position upstream of the Asp-rich domain conservedsequence I (DDXX(XX)D) (SEQ ID NO: 9) at the amino terminal side isinvolved in the control of the chain length of reaction products.

Therefore, the present invention provides a mutant prenyl diphosphatesynthase wherein an amino acid residue located at the fifth position inthe N-terminal direction from D of the N-terminal of the Asp-rich domainDDXX(XX)D (SEQ ID NO: 9) (the two X's in the parentheses may not bepresent) present in the second region of the conserved regions of theoriginal prenyl diphosphate synthase has been substituted by anotheramino acid.

The present invention also provides a DNA or an RNA encoding saidenzyme.

The present invention further provides a recombinant vector comprisingsaid DNA, specifically an expression vector.

The present invention further provides a host transformed by the abovevector.

The present invention further provides a method for producing prenyldiphosphates having 20 carbons or more characterized in that the aboveenzyme is contacted with a substrate selected from the group consistingof isopentenyl diphosphate, dimethylallyl diphosphate, geranyldiphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate.

The present invention further provides a method for producing the enzymeas set forth in any of claims 1 to 4, said method comprising culturingthe above-mentioned host and then harvesting the expression product fromthe culture.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a graph showing the enzymatic activity of the 19 mutant typeBstFPSs (B. stearothermophilus FPP synthase) obtained and a wild typeBstFPS (sample name Y). “primer: DMAPP” indicates that DMAPP was used asthe allylic substrate, “primer: GPP” indicates that GPP was used as theallylic substrate, and “primer: FPP” indicates that FPP was used as theallylic substrate. In the samples named A to W, the name of amino acidafter introduction of substitution at position 81 is indicated by aone-letter code.

FIG. 2 shows a photograph of a development pattern of TLC of thedephosphorylated product of the mutant BstFPSs' reaction when DMAPP wasused as the allylic substrate. Y81A to Y81Y represent amino acidsubstitution mutations.

FIG. 3 shows a photograph of a development pattern of TLC of thedephosphorylated product of the mutant BstFPSs' reaction when GPP wasused as the allylic substrate. Y81A to Y81Y represent amino acidsubstitution mutations.

FIG. 4 shows a photograph of a development pattern of TLC of thedephosphorylated product of the mutant BstFPSs' reaction when EPP wasused as the allylic substrate. Y81A to Y81Y represent amino acidsubstitution mutations.

FIG. 5 is a graph showing the relationship between the enzymaticactivity when DMAPP was used as the allylic substrate and the molecularweights of the amino acid side chains.

FIG. 6 is a graph showing the relationship between the enzymaticactivity when GPP was used as the allylic substrate and the molecularweights of the amino acid side chains.

FIG. 7 is a graph showing the relationship between the enzymaticactivity when FPP was used as the allylic substrate and the molecularweights of the amino acid side chains.

FIG. 8 is a graph showing the relationship between average chain lengthof the reaction products when DAMPP was used as the allylic substrateand the molecular weights of the amino acid side chains.

FIG. 9 is a graph showing the relationship between average chain lengthof the reaction products when FPP was used as the allylic substrate andthe molecular weights of the amino acid side chains.

FIG. 10 is a graph showing the relationship between average chain lengthof the reaction products when FPP was used as the allylic substrate andthe molecular weights of the amino acid side chains.

FIG. 11 is a graph showing the regions (I) to (VII) of various prenyldiphosphate synthases and Asp-rich domains, and the amino acid(asterisk) positioned at the fifth position in the N-terminal directionfrom the end thereof. In the figure, the sequence represents the aminoacid sequence of farnesyl diphosphate synthase, 1 is the one derivedfrom Bacillus stearothermophilus, 2 from Escherichia coli, 3 fromSaccharomyces cereviceae, 4 from a rat, and 5 from a human.

DETAILED DESCRIPTION

It has been proposed that there are seven conserved regions in the aminoacid sequences of prenyl diphosphate synthase (one subunit in the caseof a heterodimer) (A. Chem et al., Protine Science Vol. 3, pp. 600-607,1994). It is also known that of the five conserved regions, the regionII containing an Asp-rich domain conserved region I [DDXX(XX)D] (SEQ IDNO: 9) (the two X's in the parentheses may not be present). Althoughthere is also an Asp-rich domain in region IV, the Asp-rich domain usedto specify the modified region of the amino acid sequence of the presentinvention is present in region II, said domain being termed as theaspartic acid-rich domain I as compared to the aspartic acid-rich domainII present in region VI.

As to the prenyl diphosphate synthases having the Asp-rich domain asdescribed above, there can be mentioned farnesyl diphosphate synthase,geranylgeranyl diphosphate synthase, hexaprenyl diphosphate synthase,heptaprenyl diphosphate synthase, octaprenyl diphosphate synthase,nonaprenyl diphosphate synthase, undecaprenyl diphosphate synthase, andthe like. More specific examples include farnesyl diphosphate synthaseof Bacillus stearothermophilus, farnesyl diphosphate synthase ofEscherichia coli, farnesyl diphosphate synthase of Saccharomycescereviceae, farnesyl diphosphate synthase of the rat, farnesyldiphosphate synthase of the human, geranylgeranyl diphosphate synthaseof Neurospora crassa, hexprenyl diphosphate synthase of Saccharomycescereviceae, and the like.

By way of example of some of these, regions I to VII and the Asp-richdomain I (in the box) in region II of the amino acid sequence offarnesyl diphosphate synthases are shown in FIG. 11.

The present invention can be applicable to the prenyl diphosphatesynthases having these aspartic acid-rich domains I.

According to the present invention, the amino acid residue located atthe fifth position in the N-terminal direction from the amino acid D ofthe N-terminal of the amino acid sequence constituting said Asp-richdomain I “DDXX(XX)D” (SEQ ID NO: 9) (the two X's in the parentheses maynot be present) is substituted by another amino acid. This amino acid isindicated by an asterisk in FIG. 11. The amino acid after substitutionmay be any naturally occurring amino acid other than the original aminoacid. As one such example there is mentioned an enzyme having the aminoacid sequence in which amino acid tyrosine at the position 81 in SEQ IDNo: 1 has been substituted by a naturally occurring amino acid.

Many mutant prenyl diphosphate synthases of the present invention cansynthesize a prenyl diphosphate having a longer chain length than thatsynthesized by the native prenyl diphosphate synthase. For example, someof the farnesyl diphosphate synthases that can synthesize a farnesyldiphosphate having 15 carbons, when modified into a mutant enzyme, cansynthesize hexaprenyl diphosphate having 30 carbons.

It is known that an enzyme may retain its original enzymatic activityeven when its original amino acid sequence is modified by addition,deletion, and/or substitution of one or a few amino acids. Therefore,the present invention is intended to encompass, in addition to thepeptides having the amino acid sequence as set forth in SEQ ID No: 1,those enzymes that contain amino acid sequences modified bysubstitution, deletion, and/or addition of one or a few, for example upto 5, or up to 10, amino acids, and that can perform its originalfunction.

The present invention also provides the genes encoding variousabove-mentioned mutant enzymes, the vectors containing those genes,specifically expression vectors, and the hosts transformed with saidvectors. The gene (DNA) of the present invention can be readilyobtained, for example, by introducing mutation into the DNA encoding thenative amino acid sequence as set forth in SEQ ID No: 1 usingsite-specific mutagenesis or other conventional methods such as PCR andthe like.

Furthermore, once the amino acid sequence of the desired enzyme has beendetermined, an appropriate nucleotide sequence thereof can be determinedand the DNA can be chemically synthesized in accordance with aconventional method of DNA synthesis.

The present invention further provides an expression vector comprisingDNA such as the one mentioned above, the host transformed with saidexpression vector, and a method for producing the enzyme or peptide ofthe present invention using these hosts.

Expression vectors contain an origin of replication, expressionregulatory sequences etc., but they may differ with the hosts. As to thehosts, there can be mentioned procaryotes, for example, bacteria such asEscherichia coli, and genus Bacillus such as Bacillus subtilis, as wellas eucaryotes, for example, fungi such as yeast, for example genusSaccharomyces, such as Saccharomyces cereviceae, genus Pichia such asPichia Dastoris, filamentous fungi, for example genus Asperaillus suchas Asperaillus orvzae and Aspergillus niger, animal cells, for examplethe cultured cell of the silkworm, cultured cells of higher animals suchas CHO cell, and the like. Furthermore, plants may be used as the host.

As shown in Examples, in accordance with the present invention duringculturing the host transformed by the DNA of the present invention,long-chain prenyl diphosphates such as GGPP, GFPP, Hexpp, and the likemay be accumulated in the culture medium, which may be recovered toproduce their respective diphosphates. Furthermore, in accordance withthe invention, long-chain prenyl diphosphates may also be produced bybringing the mutant prenyl diphosphate synthase produced in accordancewith the invention in contact with a substrate isopentenyl diphosphateand allyl substrate such as farnesyl diphosphate.

When Escherichia coli is used as the host, it is known that the host hasthe regulatory functions of the gene at the stage of transcribing mRNAfrom DNA and of translating protein from mRNA. As the promoter sequenceregulating mRNA synthesis, there are known, in addition to the naturallyoccurring sequences (for example, lac, trp, bla, lpp, P_(L), P_(R), ter,T3, T7, etc.), their mutants (for example, lacUV5), and the sequences(such as tac, trc, etc.) in which a naturally occurring promoter isartificially fused, and they can be used for the present invention.

It is known that the distance between the sequence of the ribosomebiding site (GAGG and similar sequences thereof) and the initiationcodon ATG is important as the sequence regulating the ability ofsynthesizing protein from mRNA. It is also well known that a terminator(for example, a vector containing rrnPT1 T2 commercially available fromPharmacia) that directs completion of transcription termination at the3′-end affects the efficiency of protein synthesis by a recombinant.

As to the vectors that can be used for preparation of the recombinantvectors of the present invention, various vectors may be mentioned thatare derived depending on the intended use. For example, there can bementioned pBR322, pBR327, pKK223-3, pKK233-3, pTrc99, and the likehaving a replicon derived from pMBl; pUC18, pUC19, pUC118, pUCll9,pTV118N, pTV119N, pBluescript, pHSG298, pHSG396, and the like that havebeen altered to enhance copy numbers; or pACYC177, pACYC184, and thelike that have a replicon derived from pl5A; and, furthermore, plasmidsderived from pSC101, ColEl, Rl, F factor, and the like. Furthermore,fusion protein-expressing vectors that enable easier purification suchas pGEX-2T, pGEX-3X, pMal-c2 may be used. One example of the gene usedas the starting material of the present invention is described inJapanese patent application No. 6-315572.

Furthermore, in addition to plasmids, virus vectors such as λ phage orM13 phage, or transposon may be used for introduction of genes. Withregard to the introduction of the gene into microorganisms other thanEscherichia coli, gene introduction into organisms of genus Bacillus bypHY300PLK (Takara Shuzo) is known. These vectors are described inMolecular Cloning (J. Sambrook, E. F. Fritsch, and T. Maniatis, ColdSpring Harbor Laboratory Press) and Cloning Vector (P. H. Pouwels, B. E.Enger, Valk, and W. J. Brammar, Elsevier), and catalogues of manymanufacturers.

pTrc99is particularly preferable since it has, in addition to aselectable marker of the ampicillin resistant gene, a promoter,regulatory genes such as Ptrc and lacI^(q), the sequence AGGA as theribosome binding site, rrnPT₁T₂ as the terminator, and the function ofregulating expression of the gene of FPP synthase.

Integration of the DNA fragment encoding the prenyl diphosphate synthaseand, where needed, the DNA fragment having the function of regulatingexpression of the gene of said enzyme into these vectors can beperformed by a known method using an appropriate restriction enzyme andligase. Specific examples of the plasmids thus constructed include, forexample, pTV118N-Bst FPS.

As the microorganisms used for integration of genes by such recombinantvectors, Escherichia coli and microorganisms of the genus Bacillus maybe used. Such a transformation can also be carried out using the CaCl₂method and the protoplast method as described in Molecular Cloning (J.Sambrook, E. F. Fritsch, and T. Maniatis, Cold Spring Harbor LaboratoryPress) and DNA Cloning Vol. I to III (D. M. Clover ed., IRL PRESS).

In order to produce the mutant enzyme of the present invention, a hosttransformed as above is cultured, and then said culture is subjected toany method comprising salting out, precipitation with an organicsolvent, gel chromatography, affinity chromatography, hydrophobicinteraction chromatography, ion exchange chromatography, and the like torecover and purify said enzyme.

The present invention also provides a process for producing prenyldiphosphates using the enzyme of the present invention. According tothis method, the enzyme of the present invention is reacted in a medium,particularly an aqueous medium, and then, as desired, the prenyldiphosphate is recovered from the reaction medium. As the enzyme, notonly a purified enzyme but also a crude enzyme that may be semi-purifiedto various stages, or a mixture of the cultured biomass of amicroorganism may be used. Alternatively there may be used immobilizedenzymes prepared according to the conventional method from said enzyme,crude enzyme, or product containing the enzyme.

As the substrate, there may be used prenyl diphosphates and isopentenyldiphosphates having 5 to 20, preferably 5, carbons fewer than the numberof carbons of the desired prenyl diphosphate. As the reaction medium,water or an aqueous buffer solution, for example Tris buffer orphosphate buffer and the like, may be used.

By using the system of regulating chain length of the reaction productof prenyl diphosphate synthase obtained by the present invention, theprenyl diphosphate having longer chain length, synthesis of which has sofar been possible only with the hetero-dimer type enzyme, can besynthesized using mutant prenyl diphosphate synthase of the homo-dimertype that is easier to handle. Furthermore, by modifying the amino acidresidue located five amino acids upstream of the aspartic acid-richdomain I of the corresponding subunit having the aspartic acid-richdomain of the hetero-dimer type prenyl diphosphate synthase using theabove system, creation of the mutant enzyme that synthesizes prenyldiphosphates having further longer chains can be expected.

In the claims and the specification of the present invention, amino acidresidues are expressed by the one-letter codes or three-letter codes:

A; Ala; alanine

C; Cys; cystine

D; Asp; aspartic acid

E; Glu; glutamic acid

F; Phe; phenylalanine

G; Gly; glycine

H; His; histidine

I; Ile; isoleucine

K; Lys; lysine

L; Leu; leucine

M; Met; methionine

N; Asn; asparagine

P; Prl; proline

Q; Gln; glutamine

R; Arg; arginine

S; Ser; serine

T; Thr; threonine

V; Val; valine

W; Trp; tryptophan

Y; Tyr; tyrosine

Substitution of amino acid is expressed in the order of “the amino acidresidue before substitution,” “the number of the amino acid residue,”and “the amino acid residue after substitution.” For example, themutation in which a tyrosine residue at position 81 is replaced with amethionine residue is expressed as Y81M.

EXAMPLES

The present invention is now explained with reference to specificexamples, but they must not be construed to limit the invention in anyway.

Example 1

Construction of a Plasmid Containing the Gene of FPP Synthase

The gene of FPP synthase (hereinafter referred to as BstFPS) derivedfrom Bacillus stearothermophilus was subcloned at the NcoI-HindIII siteof the plasmid vector pTV118N commercially available from Takara Shuzo.The plasmid DNA was designated as pTV118N-BstFPS. The BstFPS gene isavailable from Escherichia coli JM109 (pEX1) that was internationallydeposited on Sep. 26, 1991 with the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, ofIbalaki, Japan under the accession number of FERM BP-3581. Also, theentire nucleotide sequence of the BstFPS gene has been published inJapanese patent application 3(1991)-253788, T. Koyama et al., (1993) J.Biochem. 113:355-363, or in the genetic information data bank such asGenBank under the accession number D13293. Since Bacillusstearothermophilus is also available from various depositories ofmicroorganisms such as ATCC etc., the DNA of the gene of BstFPS regioncan be obtained by the conventional gene cloning method.

Example 2

Synthesis of the Oligonucleotides for Introducing Mutation

For introduction of mutation of the gene of FPP synthase, the followingoligonucleotides were designed and synthesized:

Primer DNA (Y81X): 5′GAT CCA TAC GNN NTC TTT GAT TCA TGA TGA TTT G3′(SEQ ID No: 2)

Primer DNA (Y81N): 5′GAT CCA TAC GAA CTC TTT GAT TCA TGA TGA TTT G3′(SEQ ID No: 3)

Primer DNA (Y81I): 5′GAT CCA TAC GAT TTC TTT GAT TCA TGA TGA TTT G3′(SEQ ID No: 4)

Primer DNA (Y81M): 5′GAT CCA TAC GAT GTC TTT GAT TCA TGA TGA TTT G3′(SEQ ID No: 5)

Primer DNA (Y81F): 5′GAT CCA TAC GTT CTC TTT GAT TCA TGA TGA TTT G3′(SEQ ID No: 6)

Primer DNA (Y81P): 5′GAT CCA TAC GCC GTC TTT GAT TCA TGA TGA TTT G3′(SEQ ID No: 7)

Primer DNA (Y81V): 5′GAT CCA TAC GGT GTC TTT GAT TCA TGA TGA TTT G3′(SEQ ID No: 8)

They are designed to newly introduce the cleavage site of therestriction enzyme BspHI (5′TCATGA3′) as well as to introduce mutationin the codon encoding the amino acid residue at position 81 of BstFPS.The introduction of the cleavage site of BspHI does not change the aminoacid sequence encoded by the BstFPS gene due to degeneracy of codons.This is used to detect the substitution-mutated plasmid by means ofagarose gel electrophoresis after digestion with BspHI, since theintroduction of mutation by substitution to the amino acid residue atposition 81 of the BstFPS gene simultaneously produces a new BspHIcleavage site.

These primer DNA's were subjected to phosphorylation at 37° C. for 30minutes in the reaction medium shown below followed by denaturation at70° C. for 10 minutes:

10 pmol/μl primer DNA 2 μl 10 × kination buffer 1 μl 10 mM ATP 1 μl HO 5μl T4 polynucleotide kinase 1 μl

in which the 10×kination buffer is 1000 mM Tris-Cl (pH 8.0), 100 mMMgCl₂, and 70 mM DTT.

Example 3

Introduction of Substitution Mutation into the Codon Corresponding tothe Amino Acid Residue at Position 81 of the BstFPS Gene

Using each primer DNA constructed in Example 2, substitution mutationwas introduced into the plasmid prepared in Example 1 in accordance withthe Kunkel method. Mutan-K kit commercially available from Takara Shuzowas used to perform the Kunkel method. The experimental procedure was asdescribed in the kit insert. The substitution mutation of the plasmidneed not be conducted by the Kunkel method. For example, the same resultcan be obtained by a method using the polymerase chain reaction (PCR).

Using Escherichia coli CJ236 in the Mutan-K kit as the host cell, asingle strand DNA was obtained in which the thymine base in plasmidpTV118N-BstFPS was replaced with deoxyuracil base.

The single stranded DNA thus obtained was used as the template in areaction in which a primer DNA for synthesizing a complementary strandwas treated in the following reaction solution at 65° C. for 15 minutesand then annealed by allowing to stand at 37° C. for 15 minutes:

Single strand DNA 0.6 pmol Annealing buffer solution 1 μl Primer DNAsolution (Example 2) 1 μl H₂O make to a final volume of 10 μl

in which the annealing buffer solution is 200 mM Tris-Cl (pH 8.0), 100mM MgCl₂, 500 mM NaCl and 10 mM DTT.

Furthermore, 25 μl of an extention buffer solution, 60 units ofEscherichia coli DNA ligase, and 1 unit of T4 DNA polymerase were addedto synthesize a complementary strand at 25° C. for 2 hours. Theextention buffer solution is 50 mM Tris-Cl (pH 8.0), 60 mM ammoniumacetate, 5 mM MgCl₂, 5 mM DTT, 1 mM NAD, and 0.5 mM dNTP.

After the reaction is over, 3 μl of 0.2 M EDTA (pH 8.0) was addedthereto and was subjected to treatment at 65° C. for 5 minutes to stopthe reaction.

Example 4

Construction of a Recombinant having a Gene in which SubstitutionMutation has been Introduced into the Codon Corresponding to the AminoAcid Residue at Position 81 of the BstFPS Qene

In accordance with Example 3, the DNA solution constructed was used totransform Escherichia coli DH5a by the CaCl₂ method. An alternativemethod such as the electroporation gives the same result.

The transformant obtained by the CaCl₂ method was plated onto the agarplate containing ampicillin, a selectable marker of transformants, andwas incubated overnight at 37° C.

Among the transformants obtained as above, those substitution-mutatedpTV118N-BstFPS plasmid that has a BspHI cleavage site in BstFPS codingregion was selected. The nucleotide sequence in the neighborhood of thecodon corresponding to the amino acid residue at position 81 of theBstFPS gene of the selected substitution mutated pTV118N-BstFPS plasmidwas determined by the dideoxy method. As a result, the pTV118N-BstFPSplasmids containing the following 19 substitution mutated BstFPS geneswere obtained:

Mutation Codon Y81A GCT Y81C TGC Y81D GAC Y81E GAA Y81F TTC Y81G GGTY81H CAC Y81I ATT Y81K AAG Y81L CTC Y81M ATG Y81N AAC Y81P CCG YB1Q CAAY81R AGG Y81S TCG Y81T ACA Y81V GTG Y81W TGG Y81Y (wild type) TAC

Example 5

Measurement of Activity of the Mutant BstFPS

Crude enzyme solutions were prepared as follows from 20 transformantscomprising 19 mutant BstFPS genes obtained in Example 4 and one wildtype BstFPS gene.

The transformant cultured overnight in the 2×LB medium was centrifugedto harvest cells, and then the cells were suspended into the buffer forcell homogenization (50 mM Tris-Cl (pH 8.0), 10 mM β-meracptoethanol, 1mM EDTA). This was homogenized by sonication and then centrifuged at 4°C. at 10,000 r.p.m. for 10 minutes. The supernatant was treated at 55°C. for 30 minutes to inactivate the activity of prenyl diphosphatesynthase derived from Escherichia coli. This was further centrifugedunder the same condition and the supernatant obtained was used as acrude enzyme extract in the reaction of 55° C. for 15 minutes in thefollowing reaction solution:

[1-¹⁴C]-IPP (1 Ci/mol) 25 nmol Allylic substrate (DMAPP or GPP or FPP)25 nmol Tris-Cl (pH 8.5) 50 mM MgCl₂  5 mM NH₄Cl 50 mM β-mercaptoethanol59 mM Enzyme solution 50 μg H₂O to make 1 ml

After the reaction is over, 3 ml of butanol is added to extract thereaction product into a butanol layer. One ml of the butanol layerobtained was added into 3 ml of liquid scintillator to measureradioactivity by a scintillation counter. The result is shown in FIG. 1.Y81P mutant BstFPS has exhibited very little enzymatic activity, whichis inferably due to the fact that only the proline amino residue isderived from the imino acid, and therefore it is unable to take the formof α-helix or β-sheet structure, thereby significantly changing theessential higher structure itself of the enzyme.

The solvent is evaporated from remainder of the butanol layer by purgingnitrogen gas thereinto while heating the layer to concentrate to 0.5 ml.To the concentrate were added two ml of ethanol and one ml of potatoacid phosphatase solution (2 mg/ml potato acid phosphatase, 0.5 M sodiumacetate (pH 4.7)) to effect the dephosphorylation reaction at 37° C.Subsequently dephosphorylated reaction product was extracted with 3 mlof n-pentane. This was concentrated by evaporating the solvent bypurging nitrogen gas thereinto, which was then analyzed by TLC (reversephase TLC plate: LKC18 (Whatman), development solvent: acetone/water=9/1). The developed dephosphorylated reaction product was analyzed bythe Bio Image Analyzer BAS2000 (Fuji Photo Film) to determine thelocation and the relative radioactivity. When the amount ratio of allthe reaction products is identical, the ratio of radioactivity becomesFPP:GGPP:GFPP:HexPP=2:3:4:5. The result when DMAPP was used as theallylic substrate is shown in FIG. 2, when GPP was used as the allylicsubstrate in FIG. 3, and when FPP was used as the allylic substrate inFIG. 4.

Example 6

Relation of the Substitution-mutated Amino Acid Residue and Chain Lengthof the Reaction Product

FIG. 1 and FIG. 4 show that when the reaction was carried out using FPPas the allylic substrate most of the mutant BstFPSs converts IPP toprenyl diphosphates having chain length longer than GGPP. At this time,the substitution mutants in which the side chains of the amino acids aresuch small molecules as glycine, alanine, and serine have a higheractivity, whereas the substitution mutants in which the side chains ofthe amino acids are such large wild type molecules as tyrosine andtryptophan show a lower activity.

Then, the enzymatic activity were plotted against the molecular weightsof the side chains (FIG. 5, FIG. 6, and FIG. 7) with regard to the aminoacid residue at position 81. However, the Y81P substitution mutantenzyme in which enzymatic function was lost is excluded.

As a result, it was clearly shown that when the molecular weights of theside chains are small the activity tends to increase (FIG. 7). Thetendency was also observed even when parameters other than the molecularweight of the side chain that represents the size of the amino acidresidue was used, such as the accessible surface area i.e. a parameterof the exposed surface area of the amino acid residue [C. Chothia (1976)J. Mol. Biol. 195: 1-14, B. Lee and F. M. Richads (1971) J. Mol. Biol.55: 379-400, S. Miller et al. (1987) J. Mol. Biol. 196: 641) and thelike.

There have been very few reports so far indicating that the chain lengthof the reaction product was changed in the study on the mechanism ofcatalysis of FPP synthase by the introduction of site specific mutationwithout screening such as introduction of random mutation. The fact thatthe introduction of a single site-specific mutation enables such adynamic control of the chain length of the reaction product as obtainedby the present invention was completely unexpected.

From FIG. 5 and 6, it can be seen that when DMAPP and GPP were used asthe allylic substrate there was no significant relation between themolecular weight of the substitution-mutated amino acid residue and theenzymatic activity. This is believed to be caused by the fact that whenFPP is used as the allylic substrate the reaction specificity of thewild type enzyme is directly reflected as the enzymatic activity. Thespecificity that uses DMAPP and GPP as the allylic substrate isinherently owned by the wild type enzyme, and therefore the analysis oftendency is difficult by the parameter of enzymatic activity alone.

Therefore, the expected value of chain length of the reaction product,that is the average chain length was obtained by the following formula:

(the expected value of chain length of a reaction product)=(ratio ofFPP)×15+(ratio of GGPP)×20+(ratio of GFPP)×25+(ratio of HexPP)×30

The expected values obtained of the chain lengths of the reactionproducts were plotted against the molecular weights of the side chainsof the amino acids at position 81. However, the Y81P substitution mutantenzyme in which enzymatic function was lost is excluded. It was foundfrom these figures that the expected values of the chain length of thereaction product become higher as the molecular weight of the side chainof the amino acid residue at position 81 becomes smaller even when theallylic substrate is DMAPP or GPP.

When a similar plot analysis is made using another property of the aminoacid residue at position 81, such as Hopp & Woods Scale as a parameterof hydrophobicity [J. E. Coligan et al. (1995) Current Protocols inProtein Science, Johen Wiley & Sons, Inc.] no regular tendency isobserved as to the expected value of chain length of the reactionproduct or the enzymatic activity when FPP is used as the allylicsubstrate. Furthermore, even when parameters such as the ease of takingthe a helix structure [J. E. Coligan et al. (1995) Current Protocols inProtein Science, Johen Wiley & Sons, Inc.] or the ease of taking theβ-sheet structure [J. E. Coligan et al. (1995) Current Protocols inProtein Science, Johen Wiley & Sons, Inc.] are used, no clear relationsare observed with regard to the expected value of the chain length ofthe reaction product or the enzymatic activity when FPP was used as theallylic substrate. It was clarified for the first time by the presentinvention that the factor responsible for determining the chain lengthof the reaction product is the size of the side chain of the amino acidresidue located 5 amino acid residues upstream of the aspartic acid-richdomain I (DDXX(XX)D) (SEQ ID NO: 9).

9 1 894 DNA Bacillus stearothermophilus CDS (1)..(891) 1 gtg gcg cag ctttca gtt gaa cag ttt ctc aac gag caa aaa cag gcg 48 Met Ala Gln Leu SerVal Glu Gln Phe Leu Asn Glu Gln Lys Gln Ala 1 5 10 15 gtg gaa aca gcgctc tcc cgt tat ata gag cgc tta gaa ggg ccg gcg 96 Val Glu Thr Ala LeuSer Arg Tyr Ile Glu Arg Leu Glu Gly Pro Ala 20 25 30 aag ctg aaa aag gcgatg gcg tac tca ttg gag gcc ggc ggc aaa cga 144 Lys Leu Lys Lys Ala MetAla Tyr Ser Leu Glu Ala Gly Gly Lys Arg 35 40 45 atc cgt ccg ttg ctg cttctg tcc acc gtt cgg gcg ctc ggc aaa gac 192 Ile Arg Pro Leu Leu Leu LeuSer Thr Val Arg Ala Leu Gly Lys Asp 50 55 60 ccg gcg gtc gga ttg ccc gtcgcc tgc gcg att gaa atg atc cat acg 240 Pro Ala Val Gly Leu Pro Val AlaCys Ala Ile Glu Met Ile His Thr 65 70 75 80 tac tct ttg atc cat gat gatttg ccg agc atg gac aac gat gat ttg 288 Tyr Ser Leu Ile His Asp Asp LeuPro Ser Met Asp Asn Asp Asp Leu 85 90 95 cgg cgc ggc aag ccg acg aac cataaa gtg ttc ggc gag gcg atg gcc 336 Arg Arg Gly Lys Pro Thr Asn His LysVal Phe Gly Glu Ala Met Ala 100 105 110 atc ttg gcg ggg gac ggg ttg ttgacg tac gcg ttt caa ttg atc acc 384 Ile Leu Ala Gly Asp Gly Leu Leu ThrTyr Ala Phe Gln Leu Ile Thr 115 120 125 gaa atc gac gat gag cgc atc cctcct tcc gtc cgg ctt cgg ctc atc 432 Glu Ile Asp Asp Glu Arg Ile Pro ProSer Val Arg Leu Arg Leu Ile 130 135 140 gaa cgg ctg gcg aaa gcg gcc ggtccg gaa ggg atg gtc gcc ggt cag 480 Glu Arg Leu Ala Lys Ala Ala Gly ProGlu Gly Met Val Ala Gly Gln 145 150 155 160 gca gcc gat atg gaa gga gagggg aaa acg ctg acg ctt tcg gag ctc 528 Ala Ala Asp Met Glu Gly Glu GlyLys Thr Leu Thr Leu Ser Glu Leu 165 170 175 gaa tac att cat cgg cat aaaacc ggg aaa atg ctg caa tac agc gtg 576 Glu Tyr Ile His Arg His Lys ThrGly Lys Met Leu Gln Tyr Ser Val 180 185 190 cac gcc ggc gcc ttg atc ggcggc gct gat gcc cgg caa acg cgg gag 624 His Ala Gly Ala Leu Ile Gly GlyAla Asp Ala Arg Gln Thr Arg Glu 195 200 205 ctt gac gaa ttc gcc gcc catcta ggc ctt gcc ttt caa att cgc gat 672 Leu Asp Glu Phe Ala Ala His LeuGly Leu Ala Phe Gln Ile Arg Asp 210 215 220 gat att ctc gat att gaa ggggca gaa gaa aaa atc ggc aag ccg gtc 720 Asp Ile Leu Asp Ile Glu Gly AlaGlu Glu Lys Ile Gly Lys Pro Val 225 230 235 240 ggc agc gac caa agc aacaac aaa gcg acg tat cca gcg ttg ctg tcg 768 Gly Ser Asp Gln Ser Asn AsnLys Ala Thr Tyr Pro Ala Leu Leu Ser 245 250 255 ctt gcc ggc gcg aag gaaaag ttg gcg ttc cat atc gag gcg gcg cag 816 Leu Ala Gly Ala Lys Glu LysLeu Ala Phe His Ile Glu Ala Ala Gln 260 265 270 cgc cat tta cgg aac gccgac gtt gac ggc gcc gcg ctc gcc tat att 864 Arg His Leu Arg Asn Ala AspVal Asp Gly Ala Ala Leu Ala Tyr Ile 275 280 285 tgc gaa ctg gtc gcc gcccgc gac cat taa 894 Cys Glu Leu Val Ala Ala Arg Asp His 290 295 2 297PRT Bacillus stearothermophilus 2 Met Ala Gln Leu Ser Val Glu Gln PheLeu Asn Glu Gln Lys Gln Ala 1 5 10 15 Val Glu Thr Ala Leu Ser Arg TyrIle Glu Arg Leu Glu Gly Pro Ala 20 25 30 Lys Leu Lys Lys Ala Met Ala TyrSer Leu Glu Ala Gly Gly Lys Arg 35 40 45 Ile Arg Pro Leu Leu Leu Leu SerThr Val Arg Ala Leu Gly Lys Asp 50 55 60 Pro Ala Val Gly Leu Pro Val AlaCys Ala Ile Glu Met Ile His Thr 65 70 75 80 Tyr Ser Leu Ile His Asp AspLeu Pro Ser Met Asp Asn Asp Asp Leu 85 90 95 Arg Arg Gly Lys Pro Thr AsnHis Lys Val Phe Gly Glu Ala Met Ala 100 105 110 Ile Leu Ala Gly Asp GlyLeu Leu Thr Tyr Ala Phe Gln Leu Ile Thr 115 120 125 Glu Ile Asp Asp GluArg Ile Pro Pro Ser Val Arg Leu Arg Leu Ile 130 135 140 Glu Arg Leu AlaLys Ala Ala Gly Pro Glu Gly Met Val Ala Gly Gln 145 150 155 160 Ala AlaAsp Met Glu Gly Glu Gly Lys Thr Leu Thr Leu Ser Glu Leu 165 170 175 GluTyr Ile His Arg His Lys Thr Gly Lys Met Leu Gln Tyr Ser Val 180 185 190His Ala Gly Ala Leu Ile Gly Gly Ala Asp Ala Arg Gln Thr Arg Glu 195 200205 Leu Asp Glu Phe Ala Ala His Leu Gly Leu Ala Phe Gln Ile Arg Asp 210215 220 Asp Ile Leu Asp Ile Glu Gly Ala Glu Glu Lys Ile Gly Lys Pro Val225 230 235 240 Gly Ser Asp Gln Ser Asn Asn Lys Ala Thr Tyr Pro Ala LeuLeu Ser 245 250 255 Leu Ala Gly Ala Lys Glu Lys Leu Ala Phe His Ile GluAla Ala Gln 260 265 270 Arg His Leu Arg Asn Ala Asp Val Asp Gly Ala AlaLeu Ala Tyr Ile 275 280 285 Cys Glu Leu Val Ala Ala Arg Asp His 290 2953 34 DNA Artificial Sequence Description of Artificial Sequencesynthetic DNA 3 gatccatacg nnntctttga ttcatgatga tttg 34 4 34 DNAArtificial Sequence Description of Artificial Sequence synthetic DNA 4gatccatacg aactctttga ttcatgatga tttg 34 5 34 DNA Artificial SequenceDescription of Artificial Sequence synthetic DNA 5 gatccatacg atttctttgattcatgatga tttg 34 6 34 DNA Artificial Sequence Description ofArtificial Sequence synthetic DNA 6 gatccatacg atgtctttga ttcatgatgatttg 34 7 34 DNA Artificial Sequence Description of Artificial Sequencesynthetic DNA 7 gatccatacg ttctctttga ttcatgatga tttg 34 8 34 DNAArtificial Sequence Description of Artificial Sequence synthetic DNA 8gatccatacg ccgtctttga ttcatgatga tttg 34 9 34 DNA Artificial SequenceDescription of Artificial Sequence synthetic DNA 9 gatccatacg gtgtctttgattcatgatga tttg 34

What is claimed is:
 1. A mutant prenyl diphosphate synthase having theamino acid sequence wherein the amino acid residue Tyr at position 81 ofSEQ ID NO: 1 is replaced with an amino acid selected from the groupconsisting of Gly, Ala, Ser and Met.
 2. An enzyme according to claim 1wherein said mutant prenyl diphoshate sythase is farnesyl diphosphatesynthase, geranylgeranyl diphosphate synthase, geranylfarnesyldiphosphate synthase, or hexaprenyl diphosphate synthase.
 3. An enzymeaccording to claim 1 wherein said mutant prenyl diphosphate synthase isa thermostable enzyme.
 4. An enzyme according to claim 2 wherein saidprenyl diphosphate synthase is a thermostable enzyme.
 5. A process forproducing a prenyl diphosphate having at least 20 carbon atoms, saidprocess comprising contacting an enzyme according to any one of claims 1to 3, or 4 with a substrate selected from the group consisting ofisopentyl diphosphate, dimethylallyl diphosphate, geranyl diphosphate,farnesyl diphosphate, geranylgeranyl diphosphate.